1. Field of the Invention
The present invention relates to a protein which is a component of bromelain. In particular, the invention relates to a protein which is responsible for the anti-cancer activity of bromelain and which also has activity as an immunostimulant, and an antimicrobial agent.
2. Related Art
Bromelain is the collective name for the proteolytic enzymes found in the tissues of the plant Bromeliaceae. Although fruit bromelain is known, the most common form of bromelain is a mixture of various moieties derived from the stem of the pineapple plant (Ananas comosus). Stem bromelain (hereafter called bromelain) is known to contain at least five proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are present.
Bromelain has previously been used in the treatment of a variety of conditions including inflammation and, in particular, it has been used in the treatment of diarrhoea. The use of bromelain in the treatment of infectious diarrhoea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506, which teaches that bromelain detaches pathogens from intestinal receptors.
Taussig et al, Planta Medica, 1985, 538-539 and Maurer et al, Planta Medica, 1988, 377-381 both suggest that bromelain may be of use in inhibiting tumour growth. U.S. Pat. No. 5,223,406, DE-A-4302060 and JP-A-59225122 also teach the use of bromelain in the treatment of cancer. U.S. Pat. No. 5,223,406 teaches that bromelain is capable of inducing tumour necrosis factor (TNF) while DE-A4302060 teaches that bromelain can prevent metastasis by the structural modification of the tumour surface protein CD44.
In WO-A-9400147, various experiments were described which demonstrate that proteolytic enzymes and, in particular, bromelain, are capable of inhibiting secretion. The application also discloses that bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST). These observations were explained by the fact that one component of the bromelain mixture, stem bromelain protease, appears to be capable of modulating cyclic nucleotide pathways and this is discussed further in WO-A-9500169. In addition, bromelain has also been demonstrated to inhibit secretion caused by the calcium dependent pathway.
WO-A-9600082 also relates to bromelain and discloses that crude bromelain is capable of interfering with signalling pathways which are important for growth, in particular, signalling pathways which lead to the production of growth factors such as interleukin-2 (IL-2), platelet derived growth factor (PDGF) and insulin like growth factor (IGF). This document teaches that, as a consequence of its ability to block signalling pathways, bromelain is capable of acting as an anti-cancer agent. In addition, bromelain can be used either as an immunosuppressive agent or an immunostimulant depending on the type of cell being treated and whether the cell has previously been activated.
From the prior art, it is clear that bromelain is a mixture which has a variety of different physiological effects. Not all of the components of the bromelain mixture have been characterised and so, except for stem bromelain protease, whose activity we have described, it is not clear which of the components is responsible for which of the various different effects of bromelain. This is, of course, a major disadvantage if the bromelain mixture is to be administered as a pharmaceutical because while one component of bromelain might give the desired effect, there may well be unwanted side effects arising from the action of some other component of the bromelain mixture.
It would therefore be beneficial if individual components of bromelain giving rise to particular medicinal activities could be isolated and administered separately so as to lessen the possibility of side effects.
The present inventors have now isolated, purified and characterised one such component of bromelain which is distinct from other known components of the mixture and which has been found to have anti-cancer activity.
In a first aspect of the present invention there is provided a protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE, has an isoelectric point of 3.8 to 4.79 and has the amino terminal sequence:
Val Pro Gln Ser Ile Asp Trp Arg Asp Tyr Gly Ala Val Asn Glu Val Lys Asn (SEQ ID NO:1)
and, additionally, contains the following sequences:
Gly Gly Trp Glu Phe Lys (SEQ ID NO: 2)
Lys Ala Val Asn Gly (SEQ ID NO: 3)
Tyr Trp Ile Val Arg (SEQ ID NO: 4)
Asn Ser Trp Gly Ser Ser Trp Gly Glu Gly Gly Tyr Val Arg (SEQ ID NO:5)
Thr Ser Leu Asn His Ala Ile Thr Ile Ile Val Tyr (SEQ ID NO: 6)
Leu Pro Glu Phe (Gln) Pro (Gln) Val Leu Asp-Ala- (SEQ ID NO: 7)
Gly Val Ser Ser Ser Ser Gly Ala Cys Gly Ile Ala Met Ser Pro Leu-Thr- (SEQ ID NO: 8)
Gly Gly Val Phe Ser Gly Pro Ala Gly (SEQ ID NO: 9)
Asn Asn Ala Tyr (SEQ ID NO: 10)
Ser Ser Gly Thr Lys Tyr Trp-Val- (SEQ ID NO: 11);
where the bracketed amino acids represent alternatives to the preceding amino acid and a xe2x80x9c-xe2x80x9d represents an unidentified amino acid.
The protein of the present invention (designated CCX2 by the inventors) is largely responsible for the anti-cancer activity of bromelain since other known components such as stem bromelain protease, ananain, comosain, and F9 have all been found to have reduced anti-cancer activity. Also, because it is a single molecule, the protein of the invention does not have the disadvantages of multi-component mixtures when used as a pharmaceutical agent.
The protein may be isolated from the bromelain mixture by conventional methods, for example by chromatography. High performance liquid chromatograpy (HPLC) is suitable for the purpose and particularly good separation of bromelain proteins may be achieved by fast protein liquid chromatography (FPLC(trademark)) using a column packing material such as SP-sepharose. As will be described in more detail in the examples, in chromatography on SP-sepharose using a linear gradient of 0 to 0.8M sodium chloride in acetate buffer over 300 ml, the protein of the present invention was contained in the fraction represented by the second sharp peak off the column. This fraction was designated the CCX fraction. The protein of the invention was the major component of the CCX fraction and was isolated from the minor components by conventional methods which will be described in greater detail below.
Therefore, in a second aspect of the invention, there is provided a protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE and is obtainable by the following method:
i. dissolving bromelain in acetate buffer at pH 5.0;
ii. separating the components of bromelain by fast flow high performance liquid chromatography on S-sepharose eluting with a linear gradient of 0 to 0.8 M sodium chloride in acetate buffer over 300 ml;
iii. collecting the fraction corresponding to the second sharp peak off the column; and
iv. isolating the major protein from the fraction collected in (iii) by anion chromatography and hydrophobic interaction chromatography.
It is known from, for example, U.S. Pat. No. 5,223,406 and WO-A-9600082 that the bromelain mixture has anti cancer activity but it has previously been assumed that one of the known components of bromelain, probably stem bromelain protease, was responsible for this activity. It has now been found that this is not the case and that the protein of the present invention is an anti-cancer agent whereas stem bromelain protease has no anti-cancer activity.
The protein of the present invention has a high degree of homology with fruit bromelain protease, an enzyme isolated from fruit bromelain by Muta et al and submitted to the DDBJ/EMBL/GenBank databases on Aug. 28, 1997. The sequence identity is not complete, however and the two enzymes are, of course, derived from different sources. However, the high degree of homology between the proteins makes it likely that fruit bromelain protease will have similar anti-cancer activity to CCX2.
In a further aspect of the present invention there is provided fruit bromelain protease or an isolated and purified protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE, has an isoelectric point of 3.8 to 4.79 and has the amino terminal sequence:
Val Pro Gln Ser Ile Asp Trp Arg Asp Tyr Gly Ala Val Asn Glu Val Lys Asn (SEQ ID NO:1)
and, additionally, contains the following sequences:
Gly Gly Trp Glu Phe Lys (SEQ ID NO: 2)
Lys Ala Val Asn Gly (SEQ ID NO: 3)
Tyr Trp Ile Val Arg (SEQ ID NO: 4)
Asn Ser Trp Gly Ser Ser Trp Gly Glu Gly Gly Tyr Val Arg (SEQ ID NO:5)
Thr Ser Leu Asn His Ala Ile Thr Ile Ile Val Tyr (SEQ ID NO: 6)
Leu Pro Glu Phe (Gln) Pro (Gln) Val Leu Asp-Ala- (SEQ ID NO: 7)
Gly Val Ser Ser Ser Ser Gly Ala Cys Gly Ile Ala Met Ser Pro Leu-Thr- (SEQ ID NO: 8)
Gly Gly Val Phe Ser Gly Pro Ala Gly (SEQ ID NO: 9)
Asn Asn Ala Tyr (SEQ ID NO: 10)
Ser Ser Gly Thr Lys Tyr Trp-Val- (SEQ ID NO: 11);
where the bracketed amino acids represent alternatives to the preceding amino acid and a xe2x80x9c-xe2x80x9d represents an unidentified amino acid;
for use in human or veterinary medicine.
In particular, there is provided the protein for the treatment or prevention of cancer in a human or other mammal.
There is also provided the use of the isolated and purified protein of the first or second aspects of the invention, or fruit bromelain protease in the preparation of an anti-cancer agent.
As a result of this anti-cancer activity, the CCX2 protein or fruit bromelain protease may be used in a method for the treatment of cancer, the method comprising administering to a patient an effective amount of the isolated and purified protein of the first aspect of the invention.
It is known that the bromelain mixture has anti-cancer activity and, as discussed in our earlier application WO-A-9500169, this appears to arise from bromelain""s ability to affect intracellular signalling pathways, in particular, pathways which are modulated by MAP kinases. It is therefore possible that this is the mechanism of action of the protein of the present invention. However, the present invention is not dependent upon the correctness or otherwise of this theory.
Ras proteins help relay signals from growth-factor receptors on the surface of cells to transducer molecules to stimulate cell proliferation or differentiation. Oncogenic (or mutant) ras genes produce defective ras proteins that have acquired independence from externally supplied growth factors and, at the same time, may no longer respond to external growth-inhibitory signals. Mutant ras proteins are thus persistently hyperactive and their unbridled catalytic activity has a detrimental effect on the control of cell growth. Oncogenic ras genes therefore promote cancer and tumour formation by disrupting the normal controls on cell proliferation and differentiation. Approximately 30% of human cancers have mutations in a ras gene.
One of the transducer molecules which are activated by ras are the mitogen-activated protein (MAP) kinases (also called extracellular-signal regulated kinases [ERKs]) which transduce growth signals to the nucleus. WO-A-9500169, FIGS. 2 to 6 show that bromelain can prevent activation of the MAP kinases, ERK-1 and ERK-2. Given that signals transmitted by ras can be blocked via MAP kinase, the bromelain mixture would be expected to block cancer and tumour growth and it is possible that the protein of the present invention also works by this mechanism of action.
An alternative explanation is that the protein of the present invention acts by activating the innate immune system. The immune response has two functional divisions: the innate immune system and the adaptive immune system. The innate immune response is mediated by macrophages, natural killer cells and neutrophils. The adaptive immune response is mediated by B and T cells. When a pathogen invades the body, both the adaptive immune response and the innate immune response are activated. Innate immunity provides the first line of defence against infectious agents and most potential pathogens before they establish infection. During this initial phase of innate immunity, the adaptive immune response is developing. If the first defences are breached, the adaptive immune system should be sufficiently developed to produce a specific reaction to the infectious agent, which normally eradicates this agent. The innate immune system is also critically important in killing tumour cells.
The protein of the present invention has been shown to activate macrophages and natural killer cells (NK), critical mediators of the innate immune system important for controlling tumour growth. The protein has also been shown to increase interferon-xcex3-mediated nitric oxide (NO) production. Various publications have linked NO production to anti-tumour activity. For example, Hibbs (1991, Res. Immunol., 142, 565-569) has shown that when macrophages produce NO, they kill tumour cells in vitro. Thus, increased NO production and activation of innate immunity may be the mechanism by which the protein of the present invention acts against tumours. Again, however, the effectiveness of this protein as an anti-tumour agent is not dependent upon the correctness of this proposition.
The protein of the present invention is useful for treating many different types of cancer including solid cancers such as ovarian, colon, breast or lung cancer and melanoma as well as non-solid tumours and leukaemia.
As mentioned above, CCX2 is able to activate NK cells. NK cells are lymphocytes which can recognise and destroy cells infected with various viral, bacterial or parasitic pathogens. They are also able to kill tumour cells by specifically recognising the expression of virus-induced molecules on tumour cells or other molecules associated with tumours. Therefore, because CCX2 is able to activate NK cells, it will also be of use in the treatment or prevention of virus-induced tumours. Examples of such tumours include hepatocellular carcinoma (which may result from hepatitis B virus); non-Hodgkin""s lymphoma, nasopharyngeal carcinoma or Burkitt""s lymphoma (resulting from Epstein-Barr virus); Kaposi""s sarcoma (resulting from cytomegalovirus in HIV-infected patients); T-cell leukaemia (resulting from human T cell lymphotropic virus); and cervical carcinoma (resulting from human papilloma viruses such as HPV16 and HPV18). Again, because of the high degree of homology, fruit bromelain protease is likely to have the same activity as the CCX2 protein.
In addition to its use as an anti-tumour agent, the ability of the protein of the invention to activate the innate immune response suggests that it or fruit bromelain protease would also be of use in situations where the adaptive immune response, such as B or T cell responses, are not fully functional. This may occur in many secondary immunodeficiencies which may arise because of malnutrition, infection (for example HIV and malaria), tumours (for example lymphoid, myeloma and other), trauma (for example burns, wounds and surgery), medical treatment (for example with drugs such as steriods, cyclosporin and cyclophosphamide), protein loss (such as in diarrhoea and burns), genetic abnormalities (such as those found in combined immunodeficiency patients who lack T and/or B cells), diabetes and old age.
The present inventors have also shown that the protein of the present invention is capable of increasing interferon-xcex3-mediated NO production. Therefore, CCX2 or fruit bromelain protease may be used to treat diseases or conditions which respond to increased NO production.
NO has a critical role in host defence against infection. NO and its derivatives have potent anti-microbial activity against many pathogens including fungi, bacteria and viruses. Therefore, the protein may be administered to patients receiving chemotherapy to protect against opportunisic infections. It may also be used to treat pathogenic infections including parasites, such as Babesia, Brugia, Cryptosporidium, Encephalitoxoon, Entamoeba, Leishmania, Naegleria, Ochocerca, Opisthorchis, Plasmodium, Schistosoma, Toxoplasma and Trypanosoma. Bacteria affected by NO inlcude Bacillus, Brucella, Burkholderia, Clostridium, Ehrlichia, Francisella, Klebsiella, Legionella, Listeria, Micrococcus, Pseudomonas, Rickettsia, Salmonella, Staphylococcus, Yersinia, Chlamydia especially C. trachomatis and mycobacteria such as M. avium, M. leprae and M. tuberculosis. NO has activity against fungi such as Aspergillus, Candida, Cryptococcus, Histoplasma, Pneumocystis and Saccharomyces and against viruses, for example, Coxsackievirus, Ectomelia virus, Encephalomyocarditis virus, Epstein-Barr virus, Herpes simplex virus, Human immunodeficiency virus type 1, Japanese encephalitis virus, mouse hepatitis virus, parvovirus, poliovirus, rabies virus, simian virus 40, vaccinia virus and vesicular stomatitus virus (Fang, 1997, ASM News, 63, 668-673).
The activity of the CCX2 protein in increasing NO production complements its immunostimmulant activity and means that it can be used as an antimicrobial agent against parasites, bacteria, fungi and viruses such as those listed above.
Thus, in further aspects, the invention provides the CCX2 protein or fruit bromelain protease for use as an antimicrobial agent and the use of the CCX2 protein or fruit bromelain protease in the preparation of an antimicrobial agent.
The protein will usually be formulated before administration to patients and so, in a further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising the isolated and purified protein of the first aspect of the invention together with a pharmaceutically or veterinarily acceptable excipient.
The protein may be administered by a variety of routes including enteral, for example oral, nasal, topical, buccal, or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
In many cases, the oral route may be preferred as this is often the route which patients find most acceptable. The oral route may be particularly useful if many doses of the protein are required as will often be the case in the treatment of cancer.
When oral adminstration is chosen, it may be desirable to formulate the protein in an enteric coated preparation in order to assist its survival through the stomach. Alternatively, another orally administrable dosage form may be used, for example a syrup, elixir or a hard or soft gelatin capsule, either of which may be enteric coated.
However, if it is intended to administer only a single dose of the protein, it may be more convenient to use a parenteral route.
For parenteral adminstration, the protein may be formulated in distilled water or another pharmaceutically acceptable solvent or suspending agent.
A suitable dose of the protein to be administered to a patient may be determined by the clinician. However, as a guide, a suitable dose may be from about 0.5 to 100 mg per kg of body weight. It is expected that in most cases, the dose will be from about 1 to 50 mg per kg of body weight and preferably from 1 to 20 mg per kg of body weight. For a man having a weight of about 70 kg, a typical dose would therefore be from about 70 to 7000 mg.